Evidence for a plasma membrane-mediated permeability barrier to tat basic domain in well-differentiated epithelial cells: Lack of correlation with heparan sulfates

被引:93
作者
Violini, S
Sharma, V
Prior, JL
Dyszlewski, M
Piwnica-Worms, D
机构
[1] Washington Univ, Sch Med, Mol Imaging Ctr, Mallinckrodt Inst Radiol, St Louis, MO 63110 USA
[2] Washington Univ, Dept Mol Biol & Pharmacol, St Louis, MO 63110 USA
关键词
D O I
10.1021/bi026097e
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Membrane permeation peptides, such as Tat basic domain, have emerged as useful membrane transduction agents with potential utility in therapeutic delivery and diagnostic imaging. While generally thought to universally permeate all cells by a nonselective process, the mechanism of membrane transduction remains poorly characterized. To examine vectorial transport properties of Tat basic domain in well-differentiated epithelial cells possessing tight junctions, L and D stereoisomers of Tat(48-57) peptide conjugates labeled with Tc-99m were quantitatively analyzed in confluent monolayers of MDCK renal epithelial and CaCo-2 colonic carcinoma cells grown in transwell configurations. In both cell lines, vectorial transepithelial apparent permeability coefficients (P-app) for L- and D-[Tc-99m]Tat-peptides ranged from 30 to 70 nm/s, comparable to values for the macromolecular impermeant marker inulin in both apical-to-basolateral and basolateral-to-apical directions, but 100-fold less than the P-app values for propranolol, a highly permeable control compound. Upon direct instillation of [Tc-99m]Tat-peptide into the urinary bladder of living rats in vivo, no transepithelial permeation into other tissues was identified. Furthermore, MDCK and CaCo-2 cells showed a complete lack of intracellular accumulation of fluorescein conjugated Tat-peptide. However, translocation into cells was induced by treatment with plasma membrane permeabilizing agents such as digitonin and acetone/methanol, while cholesterol depletion with beta-methyl-cyclodextrin and metabolic inhibition with CCCP or 4 degreesC showed no effect. By contrast, in Hela and KB 3-1 cells, epithelial lines that do not form tight junctions in monolayer culture, baseline cytoplasmic and nucleolar accumulation was readily observed. Because all four cell lines expressed heparan sulfate proteoglycans, putative receptors for Tat basic peptides, we found no correlation between heparan sulfate and the permeation barrier observed in MDCK and CaCo-2 cells. The unanticipated presence of a permeation barrier to Tat-peptides in well-differentiated epithelial cells suggests the existence of cell-specific mechanisms for mediated translocation of these permeation peptides.
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页码:12652 / 12661
页数:10
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